The present invention relates to methods for the diagnosis of human autoimmune disease, for example Insulin Dependent Diabetes Mellitus (IDDM), and to methods for identifying substances which can be used in the therapy and prevention of such diseases. The invention further relates to novel human retroviruses involved in autoimmune disease and having superantigen activity, as well as to their expression products.
For some autoimmune diseases such as IDDM, Multiple Sclerosis, arthritis and others, it is known that a combination of genetic, environmental and possibly exogenous infectious factors may be important in precipitating disease. However, the precise roles of each these factors remains incompletely elucidated. For example, for IDDM, the Major Histocompatibility Complex (MHC) Class II genotype is one of the strongest genetic factors determining disease susceptibility (Vyse, T. J. and Todd J. A., 1996) although the respective roles of the different MHC Class II+ cell types in promoting disease has not yet been clarified. Furthermore, IDDM shows temporal, epidemic-like variations and the clinical disease exhibits preferential seasonal onset (Karvonen et al., 1993). Recently, Conrad et al. (1994) provided evidence for superantigen involvement in IDDM aetiology and postulated that viruses may be the modifying agent responsible for the presence of superantigen on diabetic islets.
Genetic background also has an important influence in multiple sclerosis. In addition, Perron et al (Perron et al, 1997) have recently identified a retrovirus which can be isolated from cells of multiple sclerosis patients. Whether the retrovirus contributes as a causative agent of multiple sclerosis or as a link in the pathogenic process, or whether it is merely an epiphenomenon, has not been identified. No superantigen activity of the retrovirus has been identified.
It is an aim of the present invention to identify agents implicated in the pathogenesis of human autoimmune diseases, such as IDDM, and on the basis of these agents to provide reliable diagnostic procedures and therapeutic or prophylactic substances and compositions.
These objectives are met by the provision, according to the invention, of diagnostic procedures involving the detection of expressed retroviruses having superantigen (SAg) function, these retroviruses being directly involved in the pathogenesis of human autoimmune disease by activation of autoreactive T-cells. Compounds and compositions capable of blocking SAg function or production are also provided as therapeutic and prophylactic agents in the treatment of autoimmune disease.
The present invention is based on the discovery, by the present inventors that superantigens (SAgs) encoded by retroviruses, particularly endogenous retroviruses, play a major role in the pathogenesis of autoimmune disease, very likely by activating autoreactive T-cells.
Superantigens (SAgs) (Choi et al, 1989; White et al, 1989) are microbial proteins able to mediate Interactions between MHC Class II+xe2x80x94and polyclonal T-cells resulting in reciprocal activation (Acha-Orbea et al, 1991; Choi et al, 1991; Fleischer and Schrezenmeier, 1988). Their function is restricted by only two absolute requirements: the presence of MHC Class II on the surface of the presenting cells and the expression of one or more defined Variable (V)-xcex2 T cell receptor (TCR) chain(s) on T cells.
The potential role of SAgs in human diseases is ill-defined. Bacterial SAgs have been proposed to be associated with the pathogenesis of autoimmune disease (White et al, 1989). However, although pathogen disease associations have been described, none of these have as yet implicated a pathogen-encoded SAg (Howell et al, 1991; Paliard et al, 1991). A SAg-like activity resembling the one encoded by MMTV has been reported to be associated with herpesvirus infections (Dobrescu et al, 1995; Sutkowski et al, 1996). However, in none of these two systems has it been demonstrated that the SAg activity is actually encoded by the infectious agent. SAg activity has been reported in patients having Type I diabetes (Conrad et al 1994). However, the origin of the Sag activity is not identified.
In the framework of the present invention, the inventors have identified the source of SAg activity in IDDM patients as being a novel endogenous retrovirus, (HERV) designated IDDKK1.2-22. This retrovirus is related to, but distinct from mouse mammary tumor virus (MMTV). It is ubiquitous in the human genome but is only expressed in diabetic individuals, possibly in response to a particular environmental stimulus. The HERV encodes superantigen (SAg) activity within the env gene. Expression of the SAg gives rise to preferential expansion of Vxcex2-7 T-cell receptor positive T-cells, some of which are very likely to be autoreactive. Thus the expression of self-SAg leads to systemic activation of a sub-set of T-lymphocytes, among which autoreactive T-cells, will in turn give rise to organ-specific autoimmune disease.
The involvement of retroviral SAg, particularly endogenous retroviral SAg in autoimmune disease is unexpected. Indeed, endogenous retroviruses (HERV) form an integral part of the human genome. If expressed from birth, any autoreactive T-cells activated by expression of a retroviral SAg should be deleted as part of the normal development of the immune system (thymic deletion). However, in the case of autoimmune diseases such as diabetes, the expression of the retrovirus, and hence of the encoded SAg, occurs only later in life, leading to the proliferation of autoreactive T-cells.
To identify the microbial agent responsible for SAg activity in diabetes, the present inventors have developed a novel primer-extension technique. This method can be used to isolate and identify, in a sample polyadenylated RNA, any expressed, previously unidentified retroviral RNA, particularly retroviruses having SAg activity and being involved in human autoimmune disease. This strategy relies on the following three characteristic features of functional retroviruses. First, retroviral genomes contain a primer binding site (PBS) near their 5xe2x80x2 end. Cellular tRNAs anneal to the PBS and serve as primers for Reverse Transcriptase (reviewed by Whitcomb and Hughes, 1992). Second, the R (repeat) sequence is repeated at the 5xe2x80x2 and 3xe2x80x2 ends of the viral RNA (Temin, 19B1). Third, the RT-RNAse H region of the pol gene is the most conserved sequence among different retroelements (McClure et al., 1988; Xiong and Eickbusch, 1990). The method comprises the following steps:
i) isolation of the 5xe2x80x2 R-U5 ends of expressed putative retroviral genomes using nucleic acid amplification, the 3xe2x80x2 primer being complementary to known  less than primer binding sites greater than  (pbs).
ii) isolation of the 3xe2x80x2 R-poly(A) ends corresponding to the 5xe2x80x2 R-U5 ends, by use of primers specific for the R regions isolated in step i).
iii) amplification of the conserved RT-RNase H region within the pol gene by using degenerate primers corresponding to the conserved region.
iv) amplification of the 5xe2x80x2 moiety of the putative retroviral genome by using primers specific for the different U5 regions isolated in step i) in conjunction with a primer specific for the 3xe2x80x2 end of the central pol region isolated in step iii).
v) amplification of the 3xe2x80x2 moiety of the putative retroviral genome using primers specific for the central pol region isolated in step iii) in conjunction with primers specific for the poly(A) signals present in the 3xe2x80x2 R-poly(A) sequences isolated in step ii).
vi) confirmation of the presence of an intact retroviral genome by amplification using primers specific for its predicted U5 and U3 regions.
Once an expressed retrovirus has been identified, its SAg activity can be tested by contacting a biological sample containing MHC Class II+ cells expressing the putative Sag activity, with cells bearing one or more variable (V)-xcex2 T-cell receptor (TCR) chains and detecting preferential proliferation of a Vxcex2 subset.
The techniques developed by the inventors to elucidate Sag involvement in IDDM, can be used to identify the possible involvement of expressed retrovirus and encoded SAg activity in other autoimmune diseases. The characterisation of the retrovirus and its SAg can then be made, and the particular Vxcex2-T cell receptor chain activation associated with the SAg can be identified. A given autoimmune disease can thus be defined by reference to a characterised retroviral Sag specifically associated with the disease, and to the Vxcex2-specificity or specificities. In certain autoimmune diseases, such as multiple sclerosis, it is known that T-cells with different Vxcex2 specificities can be involved in the recognition of the same immunodominant autoantigen, M.B.P. (Wucherpfennig K. W. et al, Science 1990, 25, 1016-1019). Once this  less than profile greater than  has been determined, specific diagnostic, therapeutic and prophylactic tools can be elaborated for each autoimmune disease involving retroviral SAg-stimulation of autoreactive T-cells.
The present invention involves, in a first embodiment, methods of diagnosis of autoimmune disease based on the specific expression, in autoimmune patients, of retroviruses having Sag activity.
The methods of diagnosis of the present invention are advantageous in so far as they are highly specific, distinguishing between expressed and non-expressed viral nucleic acid, and can thus be reliably used even when the pathological agent is a ubiquitous endogenous retrovirus. They can be carried out on easily accessible biological samples, such as blood or plasma, without extensive pre-treatment. The diagnostic methods of the invention detect disease-specific expression of the retrovirus and can thus be applied before appearance of clinical symptoms, for example on genetically predisposed individuals. This allows suitable therapy to be initiated before autoimmune destruction of a particular target tissue occurs.
In the context of the present invention the following terms encompass the following meanings:
a  less than human autoimmune disease greater than  is defined as a polygenic disease characterised by the selective destruction of defined tissues mediated by the immune system. Epidemiological and genetic evidence also suggests the involvement of environmental factors.
a  less than human endogenous retrovirus greater than  (HERV) is a retrovirus which is present in the form of proviral DNA integrated into the genome of all normal cells and is transmitted by Mendelian inhertance patterns. Such proviruses are products of rare infection and integration events of the retrovirus under consideration into germ cells of the ancestors of the host. Most endogenous retroviruses are transcriptionally silent or defective, but may be activated under certain conditions. Expression of the HERV may range from transcription of selected viral genes to production of complete viral particles, which may be infectious or non-infectious. Indeed, variants of HERV viruses may arise which are capable of an exogenous viral replication cycle, although direct experimental evidence for an exogenous life cycle is still missing. Thus, in some cases, endogenous retroviruses may also be present as exogenous retroviruses. These variants are included in the term  less than HERV greater than  for the purposes of the invention. In the context of the invention,  less than human endogenous retrovirus greater than  includes proviral DNA corresponding to a full retrovirus as represented schematically in FIG. 2A, comprising two LTR""s, gag, pol and env, and further includes remnants or  less than scars  greater than  of such a full retrovirus which have arisen as a results of deletions in the retroviral DNA. Such remnants include fragments of the structure depicted in FIG. 2A, and have a minimal size of one LTR. Typically, the HERVs have at least one LTR, preferably two, and all or part of gag, pol or env.
a Superantigen is a substance, normally a protein, of microbial origin that binds to major histocompatibility complex (MHC) Class II molecules and stimulates T-cell, via interaction with the Vxcex2 domain of the T-cell receptor (TCR). SAgs have the particular characteristic of being able to interact with a large proportion of the T-cell repertoire, i.e. all the members of a given Vxcex2 subset or  less than family greater than , or even with more than one Vxcex2 subset, rather than with single, molecular clones from distinct Vxcex2 families as is the case with a conventional (MHC-restricted) antigen. The superantigen is said to have a mitogenic effect that is MHC Class II dependent but MHC-unrestricted. SAgs require cells that express MHC Class II for stimulation of T-cells to occur.
 less than SAg activity greater than  signifies a capacity to stimulate T-cells in an MHC-dependent but MHC-unrestricted manner. In the context of the invention, SAg activity can be detected in a functional assay by measuring either IL-2 release by activated T-cells, or proliferation of activated T-cells.
a retrovirus having SAg activity is said to be  less than associated with greater than  a given autoimmune disease when expressed retroviral RNA can be found specifically in biological samples of autoimmune patients (ie the expressed retroviral RNA is not found in individuals free of autoimmune disease). Preferably  less than associated with greater than  further signifies in this context that retroviral SAg activation of a Vxcex2 subset gives rise directly or indirectly to proliferation of autoreactive T-cells targeting tissue characteristic of the autoimmune disease. Blockage of SAg activity thus normally prevents generation of autoreactive T-cells. Disease  less than association greater than  with Sag can also be defined immunologically or genetically: immunological association means that a particular disease-associated HLA haplotype is permissive for Sag, whereas resistant haplotypes are permissive for Sag inhibition. Genetic association implies a polymorphism in either the expression pattern of Sag or in the amino acid sequence of Sag, with Sag alleles exhibiting different degree of susceptibility to the disease.
cells which  less than functionally express greater than  Sag are cells which express Sag in a manner suitable for giving rise to MHC-dependent, MHC-unrestricted T-cell stimulation in vitro or in vivo. This requires that the cell be MHC II+ or that it has been made MHC II+ by induction by agents such as IFN-xcex3.
More particularly, in a first embodiment, the present invention relates to a process for the diagnosis of a human autoimmune disease, including pre-symptomatic diagnosis, said human autoimmune disease being associated with human retrovirus having Superantigen (SAg) activity, comprising specifically detecting in a biological sample of human origin at least one of the following:
I: the mRNA of an expressed human retrovirus known to have Superantigen (SAg) activity, or fragments of such expressed retroviral mRNA, said retrovirus being associated with a given autoimmune disease, or
II: protein expressed by said retrovirus, or
III: antibodies specific to the proteins expressed by said retrovirus, or
IV: SAg activity specifically associated with the autoimmune disease.
Thus, the diagnosis of a given autoimmune disease can be made, according to the invention, by one or more of four methods (I to IV), each involving the detection of a specific aspect of the expression of a SAg-encoding retrovirus known to be associated with the autoimmune disease, particularly an endogenous retrovirus. Detection of any of the species (I) to (IV) as listed above is indicative of the presence of the autoimmune disease specifically associated with the endogenous retrovirus under consideration or of imminent onset of the disease.
Each of the four possible methods I to IV of diagnosis of human autoimmune disease will be described in detail below.
According to method I, the autoimmune disease is diagnosed by specifically detecting in a biological sample the mRNA of an expressed human retrovirus known to have SAg activity.
Specific detection of retroviral expressed mRNA is preferably carried out using nucleic acid amplification with viral specific primers which discriminate between proviral DNA and expressed RNA template. This is of particular importance when the retrovirus associated with the autoimmune disease is an endogenous retrovirus. Indeed in such cases, the proviral DNA is present in all human cells, whether or not the autoimmune disease is present. False positives would be obtained if a detection method were used which does not distinguish between proviral DNA and transcribe mRNA.
The biological sample to be used for specific mRNA detection according to the invention may be any body fluid or tissue but is preferably plasma or blood. Normally, total RNA is extracted from the sample using conventional techniques. DNAse treatment may be carried out to reduce contaminating cellular DNA.
By performing the amplification on total RNA samples, the effects of contaminating DNA are reduced but not eliminated, even after treatment by DNAse. The method of the present invention allows selective amplification of expressed viral RNA transcripts using at least one m-RNA specific primer, for example a poly-A specific primer, even in the presence of contaminating viral DNA in the sample. The poly-A specific primer is specific for the poly-A signaals present in the R-poly(A) sequences and the 3xe2x80x2 extremity of the retrovirus (see for example FIG. 2A step 5 and FIG. 2C).
It has surprisingly been found that a poly-A-specific primer having from four to 25 t""s for example 5 or 20 T""s is optimal for the purposes of the present invention.
The mRNA specific amplification requires a reverse transcriptase (RT) step, for which the poly A-specific primer is also be used.
The second primer in the PCR step is generally complementary to the U3 region. When the amplification product has a size of about 300 to 500 nucleotides, the conditions applied for the amplification (PCR) step are normally the following:
The amplified material is subjected to gel electrophoresis and hybridised with suitable probes, for example generated from the U3 region.
By performing the mRNA specific detection of the invention, the presence of a given expressed retrovirus can be reliably determined in a biological sample. For endogenous retroviruses expression generally indicates onset of the disease process. This can be detected well before the apparition of any clinical symptoms. The diagnosis of the invention can thus be used to detect onset of the disease process, enabling treatment to be administered before irreversible autoimmune attack occurs.
The invention also encompasses pro-viral specific detection of retroviral DNA, and simultaneous detection of both expressed retroviral m-RNA and proviral DNA. Details of these methods are given in FIG. 2D and 2E, and associated legends. Specific proviral DNA detection can be used on healthy biological samples to confirm the endogenous nature of the retrovirus, the assay detecting both retroviral mRNA and proviral DNA can be used as an internal standard.
According to a preferred embodiment of the invention, the autoimmune disease detected is IDDM. The present inventors have identified, a human endogenous retrovirus associated with IDDM. This novel retrovirus (called IDDMK1.2-22) has SAg activity encoded in the NH2 terminal portion of the env gene, causing preferential proliferation of Vxcex27xe2x80x94TCR chain bearing T-cells.
IDDMK1.2-22 comprises the 5xe2x80x2 LTR, 3xe2x80x2 LTR and env-encoding sequences shown in FIGS. 7A, 7B and 7C respectively, and further comprises gag-encoding sequences. The SAg portion of the env protein occurs within the sequences shown in FIG. 7D or 7G, particularly 7G.
Diagnosis of IDDM by specific detection of expressed retroviral RNA is carried out using a polyA specific probe of the type:
5xe2x80x2 TTTTTGAGTCCCCTTAGTATTTATT 3xe2x80x2 (SEQ ID NO:1)
or similar sequence specifically hybridising to the polyA region of IDDMK1.2-22 type retroviruses, having at least 90% sequence identity with the IDDMK1.2-22 and having SAg activity.
According to a second embodiment (II) of the invention, the human autoimmune disease associated with a retroviral SAg is diagnosed by specifically detecting protein expressed by the retrovirus, particularly gag, pol or env. In the case of endogenous retroviruses, the expressed proteins may be slightly different from the expected products as a result of read-through phenomena and possibly reading-frame shifts. Preferably, the expressed protein is detected in the biological sample, such as blood or plasma, using antibodies, particularly monoclonal antibodies, specific for the said protein. A Western-like procedure is particularly preferred, but other antibody-based recognition assays may be used.
In the case of IDDM, a preferred diagnostic method comprises the detection of a protein encoded by the env gene, as shown in FIG. 7C, 7D or 7G, or the pol protein shown in FIG. 7H, or the IDDMK1.2-22 GAG protein. Alternatively, proteins having at least approximately 90% homology with these proteins, or proteins arising from read-through of internal stop codons, possibly with frame-shift, particularly a xe2x88x921 frame shift, occurring immediately after the internal stop codon. Fragments of any of these proteins having at least 6, and preferably at least 10 amino acids, for example 6-20, or 10-15 amino acids, may also be detected. Preferred proteins for this type of diagnostic assay are those having SAg activity. It is also possible to detect retroviral particles when produced.
According to a third embodiment (III) of the invention, the autoimmune disease is diagnosed by detecting in a biological sample, antibodies specific for the protein expressed by the associated retrovirus.
Detection of antibodies specific for these proteins is normally carried out by use of the corresponding retroviral protein or fragments thereof having at least 6 amino-acids, preferably at least 10, for example 6-25 amino acids. The proteins are typically Gag, Pol or Env or fragments thereof and may or may not have superantigen activity. The retroviral proteins used in the detection of the specific antibodies may be recombinant proteins obtained by introducing viral DNA encoding the appropriate part of the retrovirus into eukaryotic cell and the conditions allowing the DNA to be expressed and recovering the said protein.
In the context of the present invention, the terms xe2x80x9cantibodies specific for retroviral proteinsxe2x80x9d signifies that the antibodies show no significant cross reaction with any other proteins likely to occur in the biological sample. Generally, such antibodies specifically bind to an epitope which occurs exclusively on the retroviral protein in question. The antibodies may recognize the retroviral protein having SAg activity as presented by the M.H.C class II molecule.
Detection of specific antibodies may be carried out using conventional techniques such as sandwich assays, etc. Western blotting or other antibody-based recognition system may be used.
According to the fourth embodiment of the invention, the autoimmune disease is diagnosed by detecting, in a biological sample, SAg activity specifically associated with the autoimmune disease. This is done by carrying out a functional assay in which a biological fluid sample containing MHC class II+ cells, for example Antigen Presenting Cells (APC) such as dendritic cells is contacted with cells bearing one or more variable xcex2-T-receptor chains and detecting preferential proliferation of the Vxcex2 subset characteristic of said autoimmune disease. Typically, this method of diagnosis is combined with one or more of the methods (I), (II), (III) as described earlier to maximise specificity.
The biological sample according to this variant of the invention is typically blood and necessarily contains MHC class II+ cells such as B-lymphocytes, monocytes, macrophages or dendritic cells which have the capacity to bind the superantigen and enable it to elicit its superantigen activity. MHC class II content of the biological sample may be boosted by addition of agents such as IFN-gamma.
The biological fluid sample is contacted with cells bearing the Vxcex2-T receptors belonging to a variety of different families or subsets in order to detect which of the Vxcex2 subsets is stimulated by the putative SAg, for example V-xcex22, 3, 7, 8, 9 13 and 17. Within any one V-xcex2 family it is advantageous to use V-xcex2 chains having junctional diversity in order to confirm superantigen activity rather than nominal antigen activity.
The cells bearing the V-xcex2 receptor chains may be either an unselected population of T-cells or T-cell hybridoma. If unselected T-cells are used, the diagnostic process is normally carried out in the following manner : the biological sample containing MHC Class II+ cells is contacted with the T-cells for approximately 3 days. A growth factor such as Interleukin 2 (IL-2) which selectively amplifies activated T-cells is then added. Enrichment of, a particular V-xcex2 family or families is measured using monoclonal antibodies against the TCR-xcex2-chain. Only amplified cells are thus detected. The monoclonal antibodies are generally conjugated with a detectable marker such as a fluorochrome. The assay can be made T-cell specific by use of a second antibody, anti CD3, specifically recognizing the CD3-receptor.
T-cell hybridoma bearing defined T-cell receptor may also be used in the functional or cell-based assay for SAg activity. An example of commercially available cells of this type are given in B. Fleischer et al. Infect. Immun. 64, 987-994, 1996. Such cell-lines are available from Immunotech, Marseille, France. According to this variant, activation of a particular family of V-xcex2 hybridoma leads to release of IL-2. IL2 release is therefore measured as read-out using conventional techniques. A specific example of this procedure for diabetes is illustrated in FIG. 9. The basic methodology is adapted for other autoimmune diseases by employing T-cell receptor cells of the appropriate type for that disease.
For diabetes, detection of SAg activity will normally lead to preferential proliferation of the V-xcex27 subset. For other autoimmune diseases, other V-xcex2 subsets may be proliferated.
According to another aspect of the present invention, there is provided human endogenous retroviruses having superantigen activity and being associated with human auto immune disease Such retroviruses which may be of the HERV-K family, or otherwise, are obtainable from RNA prepared from a biological sample of human origin, by carrying out the following steps:
i) isolation of the 5xe2x80x2 R-U5 ends of expressed putative retroviral genomes using nucleic acid amplification, the 3xe2x80x2 primer being complementary to known  less than primer binding sites greater than  (pb);
ii) isolation of the 3xe2x80x2 R-poly(A) ends corresponding to the 5xe2x80x2 R-U5 ends, by use of primers specific for the R regions isolated in step i);
iii) amplification of the conserved RT-RNase H region within the pol gene by using degenerate primers corresponding to the conserved region;
iv) amplification of the 5xe2x80x2 moiety of the putative retroviral genome by using primers specific for the different U5 regions isolated in step i) in conjunction with a primer specific for the 3xe2x80x2 end of the central pol region isolated in step iii);
v) amplification of the 3xe2x80x2 moiety of the putative retroviral genome using primers specific for the central pol region isolated in step iii) in conjunction with primers specific for the poly(A) signals present in the 3xe2x80x2 R-poly(A) sequences isolated in step ii);
vi) confirmation of the presence of an intact retroviral genome by amplification using primers specific for its predicted U5 and U3 regions.
A preferred human endogenous retrovirus of the invention is IDDMK 1,2 22 comprising each of the sequences illustrated in FIGS. 7A, 7B, 7C or sequences having at least 90% identity with these sequences, and further comprising GAG-encoding sequences, and sequences encoding POL as shown in FIG. 7H. This retrovirus has a size of approximately of 8.5 kb, has SAg activity encoded within the Env region as shown in FIG. 7C and 7E and gives rise to V-xcex27 specific proliferation.
The invention also relates to proviral DNA of a retrovirus having superantigen activity and being associated with an autoimmune disease. Such proviral DNA is naturally found integrated into the human genome. The proviral DNA may be obtained from a biological sample of human origin by:
i) obtaining retroviral RNA according to the method of claim 13, and further,
ii) generating a series of DNA probes from the retroviral RNA obtained in i);
iii) hybridising under stringent conditions, the probes on a genomic human DNA library;
iv) isolation of the genomic sequences hybridising with the probes.
The invention also relates to nucleic acid molecules (RNA, DNA or cDNA) comprising fragments of the retroviral RNA or DNA described above, having at least 20 nucleotides and preferably at least 40. The fragments may be specific for a given retrovirus, specific signifying a homology of less than 20% with other human or non-human retroviruses.
Preferred nucleic acid molecules of the invention encode SAg activity particularly SAg activity, responsible for the proliferation of autoreactive T-cells. If the region of the viral genome encoding the SAg activity is unknown, the particular region may be identified by:
i) transfecting expressed retroviral DNA or portions thereof into MHC Class II+ antigen presenting cells under conditions in which the viral DNA is expressed,
ii) contacting the MHC class II+ transfectants with cells bearing one or more defined (V)-xcex2 T-cell receptor chains, and
iii) determining whether the transfectant is capable of inducing preferential proliferation of a Vxcex2 subset, the capacity to induce preferential proliferation being indicative of SAg activity within the transfected DNA or portion thereof. Proliferation may be measured by determination of 3H-thymidine incorporation (see Examples methods and materials).
The nucleic acid molecule encoding SAg activity may be derived from an endogenous human retrovirus. It typically corresponds to an open reading frame of the retrovirus and may contain at least one internal stop codon or may be a synthetic mutant in which 1 or 2 nucleotides have been added or deleted to remove the stop codon and modify the reading frame.
Preferably, the nucleic acid of the invention comprises or consists of all or part of the env gene (encoding the envelope glycoprotein) of an endogenous human retrovirus associated with autoimmune disease. The envxe2x80x94encoded protein is particularly likely to have SAg activity, as exemplified by the IDDM HERV. Synthetic or recombinant nucleic acids corresponding to the env genes or fragments thereof are also within the scope of the invention.
The nucleic acid molecules of the invention may comprise ribozymes or antisense molecules to the retrovirus involved in autoimmune disease.
The invention also relates to nucleic acid molecules capable of hybridizing in stringent conditions with retroviral DNA or RNA. Typical stringent conditions are those where the combination of temperature and salt concentration chosen to be approximately 12-20xc2x0 C. below the Tm (melting temperature) of the hybrid under study.
Such nucleic acid molecules may be labelled with conventional labelling means to act as probes or, alternatively, may be used as primers in nucleic acid amplification reactions.
Preferred nucleic acid molecules of the invention are illustrated in FIGS. 7A, 7B, 7C, 7D, 7E, 7G and also encompass nucleic acid sequences encoding the POL protein shown in FIG. 7H, and the GAG protein. Sequences exhibiting at least 90% homology with any of the afore-mentioned sequences are also comprised within the invention or fragments of any of these sequences having at least 20 and preferably at least 30 nucleotides.
The Env encoding sequence shown in FIG. 7C is particularly preferred, as well as the nucleic acid encoding the Env/F-S SAg protein shown in FIGS. 7G and 7E. A preferred nucleic acid molecule is a molecule encoding the Env/F-S Sag protein wherein the first internal stop codon (shown underlined in FIG. 7C), is mutated by insertion of an extra T (at position 517 in FIG. 7G underlined) to eliminate premature translational stop, the resulting sequence being then in the correct reading frame to encode the COOH terminal extension (shown underlined in FIG. 7G). This protein arises naturally from read-through together with a xe2x88x921 frame shift, but this process is inefficient. The synthetic Txe2x80x2-inserted cDNA provides an efficient way of producing the SAg molecule shown in FIG. 7G. The single reading frame in this  less than synthetic greater than  molecule thus corresponds to two different reading frames separated by a stop codon in the natural molecule. Nucleic acid molecules encoding an HERV env and including minus 1, plus 1 frameshifts and termination suppression (0 frames are thus particularly preferred embodiments of the invention.
The invention further relates to proteins expressed by human endogenous retroviruses having SAg activity and being associated with human autoimmune Fez disease. Peptides or fragments of these proteins having at least 6 and preferably at least 10 aminoacids, for example 6-50 or 10-30 amino acids, are also included within the scope of the invention. Such proteins may be Gag, Pol or Env proteins or may be encoded by any Open Reading Frame situated elsewhere in the viral genome. These proteins may or may not present SAg activity. Particularly preferred proteins of the invention have SAg activity. Examples of SAg proteins of the invention are proteins encoded by the env gene of HERV, for example that shown in FIG. 7G.
The proteins having SAg activity may naturally result from a premature translational stop and possibly also from a translational frameshift. Endogenous retroviral ORFs typically contain a number of internal stop codons, which often render the HERV defective. It has been discovered by the present inventors that, in some cases, retroviral expression products having SAg activity result from read-through transcription of the ORF, possibly also accompanied by a reading frame shift. Consequently, the proteins exhibiting SAg activity are not, in these cases, the expected expression products of the retrovirus.
It may therefore be deduced that open reading frames of retroviruses associated with human autoimmune disease which contain at least one internal translational stop codon are among potential candidates for SAg activity. The proteins produced by premature translational stop may have an additional carboxy-terminal extension resulting from translational frame shift, for example xe2x88x921 or xe2x88x922 or +1 or +2 translational frame shift. Such a protein is illustrated in FIG. 7G. Further preferred proteins of the invention are the proteins encoded by synthetic cDNA, corresponding to the in-frame fusion of two normally different reading frames, together with mutation of the internal stop codon. These artificial open-reading frames are made by inserting or deleting one or two nucleotides in the coding sequence at the site where frame-shift occurs naturally, thus  less than correcting greater than  the reading frame and enabling efficient production of a protein which is naturally only produced very inefficiently.
Other proteins of the invention are those comprising the aminoacid sequences shown in FIG. 7D, 7F, 7H or an aminoacid sequence having at least 80% and preferably at least 90% homology with the illustrated sequences or fragments of these sequences having at least 6 and preferably at least 10 aminoacids. The proteins of the invention may be made by synthetic or recombinant techniques.
The invention also relates to antibodies capable of specifically recognizing a protein according to the invention. These antibodies are preferably monoclonal. Preferred antibodies are those which specifically recognize a retroviral protein having SAg activity and which have the capacity to block SAg activity. The capacity of the antibody to block SAg activity may be tested by introducing the antibody under test into an assay system comprising:
i) MHC Class II+ cells expressing retroviral protein having SAg activity and
ii) cells bearing Vxcex2-T cell receptor chains of the family or families specifically stimulated by the HERV SAg expressed by the MHC Class II+ cells, and determining the capacity of the substance under test to diminish or block Vxcex2-specific stimulation by the HERV Sag.
The steps described below involve the use of Sag-expressing transfectant cells such as those described in the examples, to inhibit the effect of Sag in vitro and in vivo. The example applies to the Sag expressed by the IDDM-associated HERV, as well as to other Sags, encoded by HERV associated with other autoimmune diseases, such as multiple sclerosis, and previously identified as Sag by a functional T cell activation assay as described earlier.
Mabs directed against the Sag protein (or portion of it) are generated by standard procedures used to generate antibodies against cell surface antigens. Mice are immunised with mouse cells expressing both Sag and MHC class II (such as a. Sag-transfected mouse B cell line described in the examples below). After fusion with hybridoma cell lines, supernatants are screened for the presence of anti-Sag antibodies on microtiter plates for reactivity to Sag transfectants cells, with non-transfected cells as negative controls. Only Mabs with reactivity specific for Sag expressing cells are selected.
All such Mabs, either as culture supernatants or as ascites fluid, are then tested for their ability to block the Sag activity, as assayed by the T cell assay in the presence of Sag-expressing human MHC class II positive transfectants, as described in Example 4 below. A preferred version of this assay makes use of Vxcex2-specific hybridomas as T cell targets for read out. Controls are blocking of the same assay by anti-HLA-DR Mabs, which is known to inhibit the Sag effect on T cell activation. Mabs capable of efficiently blocking the Vxcex2-specific Sag effect, when tested at several dilutions, are selected as anti-Sag blocking Mabs.
As well as monoclonal antibodies capable of inhibiting IDDM Sag, this generation and selection of anti-Sag blocking Mabs can be achieved in the case of any HERV-encoded Sag associated with other autoimmune diseases, once such a HERV-encoded Sag has been demonstrated.
Sufficient numbers of anti-Sag Mabs are screened in the functional assay to identify anti-Sag Mabs with optimal Sag blocking activity, in terms of T cell activation (see for example FIG. 9). Selected Sag blocking Mabs are then converted into their  less than humanised greater than  counterpart by standard CDR grafting methodology (a procedure performed for a fee under contract by numerous companies). A humanised anti-Sag blocking Mab, directed against the IDDM associated Sag or against any Sag encoded by another HERV associated with autoimmunity, can then be tested clinically in patients. In the case of IDDM, early diagnosed patients are selected and protection against progressive requirement for insulin therapy is followed as an index of efficacy. In the case of other autoimmune diseases, efficacy of the anti-Sag Mab is followed with reference to the relevant clinical parameters.
The invention also relates to cells transfected with and expressing human endogenous retrovirus having SAg activity and being associated with a human autoimmune disease. The cells may be preferably human cells other than the naturally occurring cells from auto-immune patients and may also include other type of eukaryotic cells such as monkey, mouse or other higher eukaryotes. The cells may be established cell-lines and are preferably MHC class II+, or MHC II+-inducible, such as xcex2-lymphocytes and monocytes. Non-human higher eukaryotic cell-lines (e.g. mouse) stably transfected with the HERV Sags of the invention (as exemplified in Example 6 below) have been found to specifically stimulate in vitro human vxcex2-T cells of the specificity normally associated with the HERV Sag in vivo. The stimulation is coreceptor independent (CD4 and CD8). This specific T-cell stimulation can also be observed in vivo upon injection of the transfectants into non-human animals. A transgenic animal model for the human autoimmune disease is therefore technically feasible. The transgenic animal is made according to conventional techniques and includes in its genome, nucleic acid encoding the HERV Sags of the invention.
A further important aspect of the invention relates to the identification of substances capable of blocking or inhibiting SAg activity. These substances are used in prophylactic and therapeutic treatment of autoimmune diseases, involving retroviral SAg activity. The invention thus concerns methods for treating or preventing autoimmune disease, for example IDDM, by administering effective amounts of substances capable of blocking Sag activity associated with expression of a human endogenous retrovirus. The substances may be antibodies, proteins, peptides, derivatives of the HERV, derivatives of the Sag or small chemical molecules. The invention also relates to pharmaceutical compositions comprising these substances in association with physiological acceptable carriers, and to methods for the preparation of medicaments for use in therapy or prevention of autoimmune disease using these substances.
Further, this aspect of the invention includes a process for identifying substances capable of blocking or inhibiting SAg activity of an endogenous retrovirus associated with autoimmune disease, comprising introducing the substance under test into an assay system comprising:
i) MHC Class II+ cells functionally expressing retroviral protein having SAg activity and;
ii) cells bearing Vxcex2-T cell receptor chains of the family or families specifically stimulated by the HERV SAg expressed by the MHC Class II+ cells, and determining the capacity of the substance under test to diminish or block Vxcex2-specific stimulation by the HERV SAg,
The cells bearing the xcex2-T cell receptors and the MHC Class II+ cells may be those described earlier. Read-out is IL-2 release.
The substances tested for inhibition or blockage of Sag activity in such screening procedures may be proteins, peptides, antibodies, small molecules, synthetic or naturally occurring, derivatives of the retroviruses themselves, etc. Small molecules may be tested in large amounts using combinatorial chemistry libraries.
The screening procedure may include an additional preliminary step for selecting substances capable of binding to retroviral protein having SAg activity. This additional screening step comprises contacting the substances under test, optionally labelled with detectable marker with the retroviral protein having SAg activity and detecting binding.
The Sags of the invention or a portion thereof may be used for the identification of low molecular weight inhibitor molecules as drug candidates.
The rational is that because HERV encoded Sags are the product of ancient infectious agents, they are not indispensable to humans and can thus be inhibited without adverse side effects
Inhibitors of Sag, as potential drug candidates, are preferably identified by a two step process:
In the first step, compatible with large scale, high throughput, screening of collections ( less than libraries greater than ) of small molecular weight molecules, the recombinant Sag protein (or portion of it) is used in a screening assay for molecules capable of simply binding to the Sag protein (= less than ligands greater than ). Such high throughput screening assays are routinely performed by companies such as Novalon Inc or Scriptgen Inc, and are based either on competition for binding of peptides to the target protein or on changes in protein conformation induced by binding of a ligand to the target protein. Such primary high throughput screening for high affinity ligands capable of binding to a target recombinant protein are available commercially, under contract, from such companies .as Novalon or Scriptgen. This screening method requires that a HERV protein with Sag activity, and knowledge of such an activity, be available.
In the second step, any low molecular weight molecule identified as described above as capable of binding to the Sag protein, is tested in the functional Sag assay consisting of human MHC class II positive Sag transfectants and responding Vxcex2-specific T cells (preferably hybridomas), as described herein. Positive control for Sag inhibition is an anti-HLA-DR Mab, known to inhibit the Sag effect. All candidate molecules are thus tested, at different concentrations, for a quantitative assessment their anti-Sag inhibitory efficacy.
This example can apply to the Sag encoded by the IDDM-associated HERV described herein, as well as to any other Sag discovered to be encoded by another HERV associated with another autoimmune disease.
This screening procedure relies upon the availability of a Sag and of a Sag functional assay according to the invention, but it otherwise relies on commercially available steps. Compounds exhibiting anti-Sag inhibitory effects are then tested for obvious toxicity and pharmacokinetcs assays, in order to determine if they represent valuable drug candidates.
Once a substance or a composition of substances has been identified which is capable of blocking or inhibiting SAg activity, its mode of action may be identified particularly its capacity to block transcription or translation of SAg encoding sequences. This capacity can be tested by carrying out a process comprising the following steps:
i) contacting the substance under test with cells expressing retroviral protein having SAg activity, as previously defined, and
ii) detecting loss of SAg protein expression using SAg protein markers such as specific, labelled anti-SAg antibodies.
The antibodies used in such a detection process are of the type described earlier.
The invention also relates to a kit for screening substances capable of blocking SAg activity of an endogenous retrovirus associated with an autoimmune disease, or of blocking transcription or translation of the retroviral SAg protein. The kit comprises:
MHC Class II+ cells transformed with and expressing retroviral SAg according to the invention;
cells bearing Vxcex2 T-cell receptor chains of the family or families specifically stimulated by the HERV SAg;
means to detect specific Vxcex2 stimulation by HERV SAg;
optionally, labelled antibodies specifically binding to the retroviral SAg.
According to a further important aspect of the invention, there is provided a protein or peptide derived from an autoimmune related retroviral SAg as previously defined wherein the protein is modified so as to be essentially devoid of SAg activity, thereby no longer being capable of significantly activating auto-reactive T-cells. Such modified proteins are however capable of generating an immune response against SAg, the immune response involving either antibodies and/or T-cells responses. The immunogenic properties of the modified proteins are thus conserved with respect with the authentic SAg.
Such modified immunogenic proteins may be obtained by a number of conventional treatments of the SAg protein, for example by denaturation, by truncation or by mutation involving deletion, insertion or replacement of aminoacids. Modified SAg proteins being essentially devoid of SAg activity but capable of generating an immune response against SAg include the truncations of the SAg protein, either at the amino or carboxyterminal, and may involve truncations of about 5-30 aminoacids at either terminal. A preferred example with respect to the IDDMK 1.2-22 SAg encoded by the Env gene illustrated in FIG. 7, particularly in FIG. 7E and FIG. 7G, are amino and carboxy terminal truncations of the protein shown in FIG. 7G, for example truncations of 5, 10, 15, 20, 25 or 30 amino acids. An example of a C-terminal truncation of the IDDMK 1.2-22 SAg protein is the protein shown in FIG. 7D, involving a truncation of 28 amino acids. The modified protein may be obtained by recombinant or synthetic techniques, or by modifying naturally occuring SAg proteins, for example by physical or chemical treatment.
These proteins are used in the framework of the invention as vaccines, both prophylactic and therapeutic, against autoimmune disease associated with retroviral SAg. The vaccines of the invention comprise an immunogenically effective amount of the immunogenic protein in association with a pharmaceutically acceptable carried and optionally an adjuvant. The use of these vaccine compositions is particularly advantageous in association with the early diagnosis of the autoimmune disease using the method of the invention. The invention also includes the use of the immunogenic proteins in the preparation of a medicament for prophylactic or therapeutic vaccination against autoimmune diseases.
The rational behind this prospective immunisation technique is that because HERV encoded Sags are the product of ancient infectious agents, they are not indispensable to humans and can thus be inhibited without adverse side effects.
Identification of suitable anti-sag vaccine proteins or peptides can be made in the following way. Modified forms of the original active Sag protein, including truncated or mutated forms, or even specific peptides derived from the Sag protein, are first tested in the functional Sag assays described above to confirm that they have lost all Sag activity (in terms of T cell activation). These modified forms of Sag are then used to immunise mice (or humans) by standard procedures and with appropriate adjuvants. Extent and efficacy of immunisation, is measured, including circulating anti-Sag antibodies. In a preferred example, eliciting a B cell immune response, by selecting B cell epitopes from the Sag protein as immunogen, is deliberately aimed at.
Successfully immunised animals are then tested for the effect of Sag in vivo by a standard assay, namely the injection of MHC class II positive Sag transfectants (such as the transfectants described in the examples below), known to induce in vivo a Vxcex2-specific T cell activation. Successful immunisation against a Sag protein is expected to result in a reduction or in a block of the in vivo Sag-induced T cell activation and proliferation in effectively immunised individuals. This procedure is referred to as anti-Sag vaccination. Immunisation against Sag can be performed in humans, for diabetes, preferably initially in the case of early diagnosed IDDM patients. Efficacy of this novel a  less than vaccination greater than  procedure is monitored by clinical outcome and by reduction of the expected requirements for insulin therapy. In the case of other. Sags, encoded by HERV associated with autoimmune diseases other than diabetes, the clinical outcome is monitored accordingly.
The vaccines of the invention can be prepared as injectables, e.g. liquid solutions or suspensions. Solid forms for solution in, or suspension in, a liquid prior to injection also can be prepared. Optionally, the preparation also can be emulsified. The active antigenic ingredient or ingredients can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Examples of suitable excipients are water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vaccine can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants such as aluminium hydroxide or muramyl dipeptide or variations thereof. In the case of peptides, coupling to larger molecules (e.g. KLH or tetanus toxoid) sometimes enhances immunogenicity. The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration includes suppositories and, in some cases, oral formulations.
The vaccines of the invention also include nucleic acid vaccines comprising nucleic acid molecules encoding the human retroviral Sag or modified forms of the SAg known to be immunogenic but no longer active as SAgs. The nucleic acid vaccines, particularly DNA vaccines, are usually administered in association with a pharmaceutically acceptable carrier as an intra-muscular injection.
The invention also relates to use of substances inhibiting either the retroviral function or the SAg function of the associated retroviruses, or Sag synthesis, in therapy for autoimmune diseases. These substances may be identified by the screening procedures described herein.
The invention further relates to methods for treatment or prevention of autoimmune, diseases comprising administering an effective amount of a substance capable of inhibiting retroviral function or a substance capable of inhibiting SAg activity or synthesis.
An examples of compounds inhibiting retroviral function is AZT. Examples of compounds or substances capable of inhibiting SAg activity are antibodies to Sag, or ribozymes or antisense molecules to the SAg-encoding nucleic acid, or small molecules identifiedby virtue of their ability to inhibit SAg.
The invention also relates to a an exploratory process for detecting human autoimmune disease associated with expression of unidentified human retrovirus Superantigen (SAg), said process comprising at least one of the following steps:
i) detecting the presence of any expressed retrovirus in a biological sample of human origin;
ii) detecting the presence of SAg activity in a biological sample of human origin containing MHC Class II+ cells.
This process can be used as a preliminary indication of the involvement of retroviral superantigens in autoimmune disease.